U.S. patent number 5,019,393 [Application Number 07/227,728] was granted by the patent office on 1991-05-28 for biocompatible substance with thromboresistance.
This patent grant is currently assigned to New England Deaconess Hospital Corporation. Invention is credited to Ralph K. Ito, Frank W. LoGerfo.
United States Patent |
5,019,393 |
Ito , et al. |
May 28, 1991 |
Biocompatible substance with thromboresistance
Abstract
Disclosed is a biocompatible, thromboresistant substance useful
for implantable and extracorporeal devices in contact with the
vascular system, and methods for producing the same. The
biocompatible, thromboresistant substance comprises a synthetic,
biocompatible material, at least one biocompatible base coat layer
adhered to at least one surface of the material, and a
thrombogenesis inhibitor immobilized on the base coat layer via a
component capable of binding the inhibitor. The thrombogenesis
inhibitor is streptokinase, urokinase, tissue plasminogen
activator, ATPase, 5'-nucleotidase, and active fragments and active
analogs thereof.
Inventors: |
Ito; Ralph K. (Quincy, MA),
LoGerfo; Frank W. (Belmont, MA) |
Assignee: |
New England Deaconess Hospital
Corporation (Boston, MA)
|
Family
ID: |
22854220 |
Appl.
No.: |
07/227,728 |
Filed: |
August 3, 1988 |
Current U.S.
Class: |
424/423; 424/422;
424/424; 424/425; 424/426; 523/112; 523/113; 530/300; 604/266;
604/890.1 |
Current CPC
Class: |
A61L
33/0029 (20130101); A61L 33/0047 (20130101) |
Current International
Class: |
A61L
33/00 (20060101); A61F 002/00 (); A61K
009/22 () |
Field of
Search: |
;424/422-426,448
;523/112,113 ;604/266,890.1 ;530/300 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Page; Thurman
Assistant Examiner: Kishore; G. S.
Attorney, Agent or Firm: Lahive & Cockfield
Claims
We claim:
1. A biocompatible substance with thromboresistance comprising:
(a) a synthetic, polymeric, biocompatible material;
(b) at least one biocompatible base coat layer adhered to at least
one surface of said material; and
(c) a thrombogenesis inhibitor immobilized on said base coat layer,
said thrombogenesis inhibitor being other than hirudin or an active
analog or fragment of hirudin,
said base coat layer having a component capable of binding said
thrombogenesis inhibitor.
2. The substance of claim 1 wherein said thrombogenesis inhibitor
is an inhibitor selected from the group consisting of
streptokinase, urokinase, tissue plasminogen activator, ATPase,
ADPase, 5'-nucleotidase, and active fragments, and active analogs
thereof, and mixtures thereof.
3. The substance of claim 1 wherein said polymer is selected from
the group consisting of Dacron, nylon, polyurethane, cross-linked
collagen, polyglycolic acid, polytetrafluoroethylene, and mixtures
thereof.
4. The substance of claim 3 wherein said polymer comprises
polyethylene terphthalate.
5. The substance of claim 1 wherein said base coat layer comprises
a component selected from the group consisting of a protein,
peptide, lipoprotein, glycoprotein, glycosaminoglycan, hydrogel,
synthetic polymer, and mixtures thereof.
6. The substance of claim 5 wherein said component of said base
coat layer comprises a protein.
7. The substance of claim 6 wherein said protein is selected from
the group consisting of serum albumin, fibronectin, and mixtures
thereof.
8. The substance of claim 7 wherein said protein comprises bovine
serum albumin.
9. The substance of claim 7 wherein said protein comprises human
serum albumin.
10. The substance of claim 7 wherein said protein comprises bovine
fibronectin.
11. The substance of claim 7 wherein said protein comprises human
fibronectin.
12. The substance of claim 1 further comprising at least one
molecule of a bifunctional cross-linking reagent linking said
thrombogenesis inhibitor to said base coat layer.
13. The substance of claim 12 wherein said bifunctional
cross-linking reagent is heterobifunctional.
14. The substance of claim 12 wherein said bifunctional
cross-linking reagent is homobifunctional.
15. The substance of claim 13 wherein said heterobifunctional
cross-linking reagent comprises SPDP.
16. A method of producing a biocompatible, thromboresistant
substance, said method comprising the steps of:
(a) adhering at least one base coat layer to at least one surface
of a synthetic, polymeric, biocompatible material, said base coat
layer containing a component capable of binding said base coat
layer thereto; and
(b) immobilizing a thrombogenesis inhibitor on said adhered base
coat layer, said thrombogenesis inhibitor being other than hirudin
or an analog or fragment of hirudin.
17. The method of claim 16 wherein said immobilizing step further
comprises immobilizing a thrombogenesis inhibitor selected from the
group consisting of streptokinase, urokinase, tissue plasminogen
activator, ATPase, ADPase, 5'-nucleotidase, and active fragments,
and active analogs thereof, and mixtures thereof.
18. The method of claim 16 wherein said adhering step
comprises:
(a) activating said material so as to enhance the binding of said
base coat layer thereto; and
(b) contacting said activated material with said base coat layer
for a time sufficient to allow said component of said base coat
layer to bind to said activated material.
19. The method of claim 18 wherein said adhering step comprises
adhering a base coat to at least one surface of a synthetic,
polymeric, biocompatible material, said base coat layer containing
a component selected from the group consisting of a protein,
peptide, lipoprotein, glycoprotein, glycosaminoglycan, hydrogel,
synthetic polymers, and mixtures thereof.
20. The method of claim 19 wherein said adhering step further
comprises adhering a base coat layer containing a protein to at
least one surface of said material.
21. The method of claim 20 wherein said adhering step further
comprises adhering a base coat layer to at least one surface of
said material, said base coat layer containing a protein selected
from the group consisting of serum albumin, fibronectin, and
mixtures thereof.
22. The method of claim 21 wherein said adhering step further
comprises adhering a base coat layer containing human serum albumin
to at least one surface of said material.
23. The method of claim 21 wherein said adhering step further
comprises adhering a base coat layer containing bovine serum
albumin to at least one surface of said material.
24. The method of claim 21 wherein said adhering step further
comprises adhering a base coat layer containing human fibronectin
to at least one surface of said material.
25. The method of claim 21 wherein said adhering step further
comprises adhering a base coat layer containing bovine fibronectin
to at least one surface of said material.
26. The method of claim 18 wherein said activating step comprises
the steps of:
(a) treating said material with a solution that makes available for
binding at least one chemically active group in said material;
and
(b) contacting said treated material with a solution containing a
bifunctional cross-linking reagent for a time sufficient to allow
binding of said group to said reagent.
27. The method of claim 26 wherein said treating step further
comprises treating said material with a solution that makes
available for binding at least one carboxylic acid group in said
material.
28. The method of claim 18 further comprising the preliminary step
of contacting said material with a solution which removes
impurities thereon, said preliminary step being performed prior to
said adhering step.
29. The method of claim 18 wherein said immobilizing step further
comprises the steps of:
(a) contacting said thrombogenesis inhibitor with a at least one
molecule of a bifunctional cross-linking reagent for a time
sufficient to allow linking of said reagent to said thrombogenesis
inhibitor; and
(b) binding said thrombogenesis inhibitor-linked reagent to said
base coat layer.
30. The method of claim 29 wherein said contacting step further
comprises contacting said base coat with at least one molecule of
said bifunctional cross-linking reagent for a time sufficient to
allow linking of said agent to said base coat,
and said binding step further includes binding said thrombogenesis
inhibitor-linked reagent to said base coat-linked reagent.
31. The method of claim 29 wherein said contacting step further
includes contacting said thrombogenesis inhibitor with at least one
molecule of said bifunctional cross-linking reagent selected from
the group consisting of heterobifunctional cross-linking reagents,
homobifunctional cross-linking reagents, and mixtures thereof.
32. The method of claim 30 wherein said contacting step includes
contacting said base coat with at least one molecule of said
bifunctional cross-linking reagent selected from the group
consisting of heterobifunctional cross-linking reagents,
homobifunctional cross-linking reagents, and mixtures thereof.
33. The method of claim 30 further comprising the steps of:
(a) reducing said base coat-linked reagent to expose a first
sulfhydryl group thereon;
(b) adding said inhibitor-linked reagent to said exposed sulfhydryl
group thereon; and
(c) inducing a substitution reaction involving said sulfhydryl
group and said inhibitor-linked reagent, said reaction resulting in
linkage of said base coat to said inhibitor.
34. The method of claim 31 wherein said contacting step includes
contacting said thrombogenesis inhibitor with the
heterobifunctional cross-linking reagent, N-succinimidyl
3-(2-pyridylaithio)propionate (SPDP).
35. The method of claim 32 wherein said contacting step includes
contacting said thrombogenesis inhibitor with the
heterobifunctional crosslinking reagent, SPDP.
36. The method of claim 29 further comprising the additional step
of subjecting said throbogenesis-linked reagent to a
chromatographic procedure to remove impurities therein, said
additional step being performed after said contacting step and
prior to said binding step.
37. A method of producing a biocompatible, thromboresistant
substance, said method comprising the steps of:
(a) immobilizing a thrombogenesis inhibitor to a base coat
layer,
said inhibitor being other than hirudin or an active analog or
fragment of hirudin, and
said base coat layer containing a component capable of binding said
thrombogenesis inhibitor; and
(b) adhering said base coat layer linked to said thrombogenesis
inhibitor to at least one surface of a synthetic, polymeric,
biocompatible material.
Description
CROSS-REFERENCE TO RELATED APPLICATION
The subject matter of this application is related to applicants'
copending application Ser. No. 227,700, entitled "HIRUDIN-COATED
BIOCOMPATIBLE SUBSTANCE", filed on even date herewith.
BACKGROUND OF THE INVENTION
The technical field of the present invention is prosthetic vascular
materials, and more specifically is biocompatible, thromboresistant
substances and methods of their preparation.
Exposure of blood to artificial surfaces usually leads to
deposition of a layer of adherent platelets, accompanied by
activation of the intrinsic coagulation system, and ultimately to
the formation of a thrombus. In fact, significant blood/materials
interaction can occur on a single pass through a prosthetic
arterial graft. The types of blood proteins initially adsorbed or
bound to synthetic surfaces may include proteins involved in
contact coagulation. Contact coagulation or the extrinsic pathway
of coagulation is a complex pathway of biochemical events that
induces fibrin formation, platelet and complement activation,
chemotaxis, kinin generation, and activation of fibrinolytic
components. In addition, each of these events augments subsequent
biochemical pathways often controlled by positive and negative
feedback loops. Thus, thrombosis induced by contact with artificial
materials is a major obstacle in the development and use of
internal prostheses and extracorporeal devices such as artificial
vessels and organs, and cardiopulmonary bypass and hemodialysis
equipment.
Materials having varying degrees of thromboresistance have been
utilized in vascular prostheses with limited success. These
materials include corroding (self-cleaning) metals, synthetic
polymers such as polydimethyl siloxane, Teflon, acylates and
methacrylates such as Dacron, electrets, anionic copolymers, and
hydrogels for a review see Salzman et al. (1987) in Hemostasis and
Thrombosis, Basic Principles and Clinical Practice (Colman et al.,
eds.) J. B. Lippincott Co., Phila., Pa., pp. 1335-1347).
To decrease the chances of thrombosis due to extended periods of
contact with such artificial materials, patients have been treated
with systemically administered anti-coagulant, anti-platelet, and
thrombolytic drugs. These include any compound which selectively
inhibits thromboxane synthetase without affecting prostacycline
synthetase, affects platelet adherence as well as aggregation and
release, enhances vascular PGI2 production, and/or inhibits both
thrombin- and thromboxane-mediated platelet aggregation. Such
compounds include aspirin, sulfinpyrazone, dipyridamole,
ticlopidine, and suloctidil. However, treatment with these drugs
often elicits unwanted side effects including systemic hemmorhaging
and the inability to initiate and complete desired clotting
elsewhere in the body.
To improve on the thromboresistance of artificial materials,
biologically active molecules having thrombolytic, anticoagulating,
thrombogenesis-inhibiting, and/or platelet inhibiting abilities
have been linked thereto. For example, heparin has been bound to
artificial surfaces to reduce cooagulation by activating variuous
inhibitors of the intrinsic clotting system (Salzman et al. (1987)
in Hemostasis and Thrombosis: Basic Principles and Clinical
Practice. 2nd Ed., (Colman et al., eds.), Lippincott Co., Phila.,
Pa., pp. 1335-1347). However, heparin enhances platelet responses
to stimuli such as ADP or collagen, and promotes two adverse
primary blood responses towards synthetic surfaces: platelet
adhesion and aggregation. In addition, although surface-bound
heparin/antithrombin complex may be passive towards platelets, the
wide variety of effects it has on interactions with endothelial
cell growth factor, inhibition of smooth muscle proliferation, and
activation of lipoprotein lipase raises questions as to what
adverse effects it may induce over time.
Anti-platelet agents such as PGE.sub.1, PGI.sub.2 (experimental use
only), cyclic AMP, and aspirin have also been attached to solid
polymer surfaces. These agents discourage the release of platelet
factors that stimulate adverse healing responses in the vicinity of
a vascular graft. They may also reduce platelet-aided thrombus
formation by inhibiting platelet adhesion.
The exposure of many artificial surfaces to albumin prior to
vascular contact results in reduced reactivity with platelets (NIH
Publication No. 85-2185, Sept., 1985, pp. 19-63). Therefore,
albumin has been used to coat extracorporeal surfaces before
cardiopulmonary by-pass surgery. However, long-term
thermoresistance has not been achieved by this procedure.
Fibrinolytically active streptokinase and urokinase, alone or in
combination with heparin have been attached to artificial surfaces
by Kusserow et al (Trans. Am. Soc. Artif. Intern. Organs (1971)
17:1). These enzymes reduce excessive fibrin deposition and/or
thrombotic occlusions. However, the long term assessment of their
ability to confer thromboresistance to a synthetic surface has not
been determined.
Surface active agents such as Pluronic F-68 have also been
immobilized on artificial surfaces, but do not appear to offer long
term blood compatibility (Salyer et al. (1971) Medical Applications
of Plastics. Biomed. Materials Res. Sym. (Gregor, ed.) No. 1 pp.
105).
Therefore, what is needed are better biocompatible materials which
are thromboresistant in the long term and whose active components
do not cause detrimental side affects.
An object of the present invention is to provide a synthetic,
biocompatible, thromboresistent material useful for implantable and
extracorporeal devices in contact with bodily fluids.
Another object is to provide an immobilized thrombogenesis
inhibitor which is biologically active, and a method of preparing
the same.
Still another object of this invention is to provide a method of
inhibiting platelet aggregation, the release of platelet factors,
and thrombogenesis at the localized site of the graft or
prosthesis-blood interface, thus avoiding the systemic effect of
antiplatelet and antithrombosis drugs.
SUMMARY OF THE INVENTION
Materials and methods are disclosed herein for the provision of
biocompatible, thromboresistant substances useful as a component of
implantable or extracorporeal devices in contact with the
blood.
It has been discovered that a synthetic, biocompatible material can
be made into a thromboresistant substance by immobilizing to it, by
way of a base coat layer, a thrombogenesis inhibitor other than
hirudin, or an active analog or fragment thereof, in such a way
that does not compromise its thrombogenesis inhibiting
activity.
The term "thrombogenesis inhibitor" is used herein to describe
molecules which interfere with or inhibit the formation of a
thrombus. Such molecules include those which interfere or inhibit
the intrinsic and extrinsic coagulation system, platelet adherence,
aggregation, or factor release or activity, or the release or
active of tissue factors. Included are native, synthetic, or
recombinant proteins, or active analogs, fragments, derivatives, or
fusion products thereof, and mixture thereof which can interfere
and inhibit the formation of a thrombus. Thrombogensis inhibitors
useful for imparting thromboresistance to the substance include
proteins which are mambrane-bound in their native state (e.g.,
adenosine triphosphatase (ATPase), adenosine diphosphatase
(ADPase), and 3'-nucleotidase), and those which are normally
soluble in vivo (e.g., tissue plasminogen activator (tPA),
urokinase (UK), and streptokinase (SK)). However, other molecules
which inhibit or interfer with the activity of other thrombogenesis
inhibitors are useful as well.
Synthetic materials contemplated by the instant invention are
preferably polymers such as Dacron, nylon, polyurethane,
cross-linked collagen, polytetrafluoroethylene, polyglycolic acid,
and mixtures thereof, the most preferred polymeric material being
Dacron. Other synthetic materials might also be used.
At least one layer of biocompatible material is adhered to at least
one surface of the synthetic material. This base coat layer
contains a component which is capable of binding the thrombogenesis
inhibitor. Examples of such base coat components include proteins,
peptides, lipoproteins, glycoproteins, glycosaminoglycans,
hydrogels, synthetic polymers, and mixtures thereof. In preferred
aspects of the invention, the base coat layer includes a protein
component such as serum albumin or fibronectin from, for example,
human or bovine sources, or mixtures of these proteins. Other
materials might also be used to form the base coat layer.
In accordance with the invention, the thrombogenesis inhibitor is
immobilized on the synthetic material via a base coat layer which
is adhered to least one surface cf the synthetic material. The base
coat layer contains a component capable of binding the
thrombogenesis inhibitor without compromising the biological
activity of the inhibitor.
In exemplary aspects of the invention, the synthetic material is
activated prior to having the base coat layer adhered thereto so as
to enhances its ability to bind the base coat base layer. For
example, in one preferred aspect, the synthetic material is
contacted with a solution which makes available at least one
chemically active group (e.g., a carboxylic acid group) in the
material for binding to a bifunctional cross-linking reagent (e.g.,
carbodiimide). The material so treated is then put into contact
with a solution containing the cross-linking carbodiimide reagent
for a time sufficient to allow the chemically active group to bind
thereto.
In another embodiment, the synthetic material may be contacted with
a solution which removes impuritities therein and/or thereon prior
to the activation step described above.
The immobilization step may be carried out by initially contacting
the thrombogenesis inhibitor with at least one molecule of a
bifunctional cross-linking reagent for a time sufficient to allow
linking of the reagent to the inhibitor, and then binding the
thrombogenesis inhibitor-linked reagent to the base coat. The bound
thrombogenesis inhibitor retains its thrombogenesis inhibiting
activity when bound to the reagent. The bifunctional cross-linking
reagent useful for such an immobilization step may be
heterobifunctional (e.g., N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP)), homobifunctional (e.g.,
ethylene glycolbis (succinimidylsuccinate) (EGS)), or a mixture of
both.
The term "bifunctional cross-linking reagent" is defined herein as
a molecule having the ability to bind to, and therefore link, two
reactive groups on, for example, one molecule or two separate
molecules. If the bifunctional cross-linking reagent binds two
different types of groups, it is a "heterobifunctional"
cross-linking reagent. However, if the bifunctional cross-linking
reagent binds only to two similar groups, it is
"homobifunctional".
Prior to the binding step, the thrombogenesis-linked reagent :nay
be subjected to chromatographic procedures to remove impurities
mixed in with it.
In an alternative aspect of the invention, the base coat adhered to
the synthetic material may be linked at the same time to at least
one molecule of a bifunctional cross-linking reagent. In this
embodiment, the method further includes binding the thrombogenesis
inhibitor-linked reagent to the base coat-linked reagent, thereby
linking the thrombogenesis inhibitor to the material-adhered base
coat layer.
In another aspect of the invention, the base coat-linked reagent is
reduced prior to the binding step. Reduction results in the
formation of sulhydryl groups from the reagent on the base coat
which can react with the inhibitor-linked bifunctional
cross-linking reagent via a substitution reaction to form an S--S
bond, thereby covalently linking the thrombogenesis inhibitor to
the base coat.
In yet another aspect of the invention, the base coat is linked to
the thrombogenesis inhibitor before it is linked to the synthetic,
biocompatible material.
The invention will next be described in connection with certain
illustrated embodiments. However, it should be clear that various
modifications, additions, and deletions can be made without
departing from the spirit or scope of the invention.
BRIEF DESCRIPTION OF THE DRAWING
The foregoing and other objects of the present invention, the
various features thereof, as well as the inventions thereof may be
more fully understood from the following description when read
together with the accompanying drawings in which:
FIG. 1 is a diagrammatic representation of the pathways involved in
thrombogenesis; and
FIG. 2 is a diagrammatic representation of platelet involvement in
thrombogenesis.
DESCRIPTION OF THE INVENTION
This invention provides biocompatible, thromboresistant substances
useful for implantable and extracorporeal devices in contact with
the vascular system, and methods for their fabrication.
The substances provided by this invention include a synthetic
biocompatible substance having a thrombogenesis-inhibiting reagent
linked thereto via a biocompatible base coat adhered to the
material's surface.
The material useful in a prosthetic extracorporeal or implantable
device may be composed of any biocompatible, synthetic, preferably
polymeric material having enough tensile strength to withstand the
rigors of blood circulation, and having groups onto which a base
coat can be directly or indirectly bound. Examples of such
synthetic materials are polytetrafluoroethylene (Teflon) and
Dacron, nylon, and the like. The material may have any dimensions
suitable for the purpose for which it is being used. For example,
it may be an integral part of an implanted heart valve or of an
extracorporeal device used for hemodialysis or cardiopulmonary
by-pass surgery, or it may be used to coat catheters or to line the
interior of a vascular graft.
The synthetic material, when obtained, may be coated with or
contain various noncovalently adhered impurities whose removal may
be prerequisite for the adherence of a base coat thereto. For
example, lubricants on commercial quality Dacron can be removed by
contacting the Dacron with a solution containing, for example,
various detergents, solvents, or salts, which loosen and/or
solubilize these impurities.
TABLEs 1 and 2 outline representative methods of preparing the
biocompatible, thromboresistant substance, where "Da" refers to a
synthetic material composed of woven Dacron fibers, and "HSA"
refers to human serum albumin.
TABLE 1 ______________________________________ STEP PROCESS
______________________________________ (1) Da. + NaOH .fwdarw.
Da-COOH (2) Da-COOH + EDC .fwdarw. Da-EDC (3) Da-EDC + HSA .fwdarw.
Da-HSA + urea (EDC by-product) (4) Da-HSA + SPDP .fwdarw.
Da-HSA-SPDP (5) Da-HSA-SPDP + DTT .fwdarw. Da-HSA-SH + P-2-T (6)
Inhibitor + SPDP .fwdarw. Inhibitor-SPDP (7) Da-HSA-SH +
Inhibitor-SPDP .fwdarw. Da-HSA-S-S-Inhibitor + P-2-T
______________________________________
TABLE 2
__________________________________________________________________________
STEP PROCESS
__________________________________________________________________________
(1) HSA + SPDP .fwdarw. HSA-SPDP (2) HSA-SPDP + DTT .fwdarw. HSA-SH
+ P-2-T (3) Inhibitor + SPDP .fwdarw. Inhibitor-SPDP (4) HSA-SH +
Inhibitor-SPDP .fwdarw. HSA-S-S-Inhibitor + P-2-T (5) Da + NaOH
.fwdarw. Da-COOH (6) Da-COOH + EDC .fwdarw. Da-EDC (7) Da-EDC +
HSA-S-S-Inhibitor .fwdarw. Da-HSA-S-S-Inhibitor + urea (EDC
by-product)
__________________________________________________________________________
Initially, the material may be activated so as to enhance the
binding of the base coat layer. This activating step increases the
number of chemically active groups in the material. For example,
alkaline hydrolysis may be performed to increase the number of
reactive carboxylic acid groups in the Dacron to which a
bifunctional cross-linking reagent such as carbodiimide may be
bound. Ultimately, the base coat will adhere to the bound
carbodiimide groups on the material. However, this method must be
performed with care, as alkaline hydrolysis partially degrades the
Dacron, resulting in a fraying of the material's fibers.
At least one base coat layer is adhered to at least one surface of
the synthetic material.
This layer, either adhered to the material or unbound, provides
components for attachment of the thrombogenesis inhibitor. Such
components provide more binding sites for the inhibitor than the
synthetic material, alone, thereby amplifying the amount of
inhibitor which may be bound. Useful components include proteins,
peptides, lipoproteins, glycoproteins, glycosaminoglycans,
synthetic polymers, and mixtures thereof. Proteins such as serum
albumin and fibronectin are particularly useful for this purpose as
they are known to have anti-thrombogenic properties, themselves,
are very desirable as base coat components (Lyman et al. (1965)
Trans. Am. Soc. Artif. Intern. Organs 11:301; Falb et al. (1971)
Fed. Proc. 30: 1688). An HSA molecule, for example, has 65 amino
groups available as binding sites.
Attachment of the base coat to the artificial surface may be
covalent in nature. Methods to covalently bind proteins to Dacron
involve attack of the free reactive succinimide ester group of the
cross-linking reagent to primary amino groups on a protein. As
shown in the example in Table 1, to covalently adhere the base coat
to Dacron, the Dacron is initially treated with 0.5 N NaOH and
reacted with carbodiimide before it is coated with HSA (base coat)
in phosphate buffered saline (PBS).
A thrombogenesis inhibitor useful as a coating for surfaces in
contact with blood, bodily fluids, or tissues, is then covalently
adhered to the base coat via the component. Inhibitor-coated
substances are ideal for implantable use in devices which are in
direct contact with blood. For example, by-pass grafts used to
replace blood vessels often become filled with blood clots or
thrombi, resulting in restricted blood flow. Since the
inhibitor-coated substance is resistant to formation of blood
clots, thrombosis and subsequent blockage of the bypass graft will
be prevented. Likewise when catheters are placed into the vascular
system for a diagnostic or therapeutic purposes, a blood clot often
forms on the outside of the catheter. The clot may be washed off
the catheter by flowing blood, or be jarred loose by manipulation
of the catheter, increasing the possibility of embolism and
blockage of the circulation to vital organs. Inhibitor-coated
substances provide similar advantages for artificial or prosthetic
heart valves, intraaortic balloon pumps, total or artificial heart
or heart assist devices, intracaval devices, and any device in
contact with the bloodstream. In addition, inhibitor-coated devices
provide advantages for intracavity devices such as intraperitoneal
dialysis catheters and subcutaneous implants where the
thrombin-induced inflammmatory reactions would be diminished.
Thrombogenesis inhibitors useful for these purposes include
molecules which interfere with, or inhibit thrombogenesis. These
include ATPase, ADPase, 5'-nucleotidase, streptokinase, urokinase,
tissue plasminogen activator, anticoagulants, platelet inhibitors
(e.g., prostacycline and aspirin), and active analogs, fragments,
derivatives, and fusion products thereof, or mixtures thereof.
ADPase reduces platelet aggregation by degrading ADP. ADP is stored
in the dense granules of platelets and can be released by thrombin,
epinephrine, collagen, and other stimulants. When released, ADP
promotes platelet receptor binding to fibrinogen and to von
Willebrand Factor, two proteins essential for aggregation, and then
promotes thromboxane synthesis, platelet aggregation, more ADP
release, and thus, self-enhances platelet aggregation.
ATPase catalyzes the conversion of ATP to ADP, which can then be
acted upon by ADPase, as described above.
5'-nucleotidase (AMPase), like ADPase, degrades ADP. It also
degrades AMP, a competitive antagonist for ADP receptor birding, to
adenosine, a platelet inhibitor. Adenosine inhibits platelet
aggregation by increasing intracellular levels of cyclic AMP. High
levels of cyclic AMP inhibit mobilization of calcium ions from
platelet storage pools. Free calcium ions within the platelet
stimulates release of additional ADP from platelet dense granules,
and is necessary for platelet aggregation, hence thrombogenesis
(see FIG. 2).
The thrombogenesis inhibitor is directly or indirectly immobilized
on the base coat via the use of a bifunctional cross-linking
reagent. In particular, a heterobifunctional cross-linking reagent
which has two different reactive groups at each end of a linear
molecule, and can therefore bind two different reactive groups on
other molecules or on a different region of the same molecule, is
most useful as a bifunctional cross-linking agent. For example,
photoreactive cross-linkers, such as sulfosuccinimidyl
2-(m-azodo-o-nitro-benzamido)ethyl-1, 3'-dithio-propionate (SAND),
or N-succinimidyl-6-(4;azoido-2'-nitrophenyl-amino) hexanoate
(SANPAH) have a photoreactive group that can directly insert into
C--H bonds of the base coat by photochemical coupling, while the
other group remains free to bind to proteins.
Other useful and preferable cross-linking reagents (such as SPDP)
and their characteristics are found in TABLE 3. In TABLE 3, the
"Double-Agent Number" listed for each reagent is the commercial
designation for the reagent as made available by Pierce Chemical
Co. (Rockford, Ill.).
TABLE 3 ______________________________________ CROSS-LINKING
REAGENTS (part A) Reactive Double- Double- towards: Agent Agent
Bifunctionality Photo- Number Acronym Homo Hetero NH.sub.2 SH
Reactive ______________________________________ 21551 ANB-NOS X X X
20106 APB X X X 20107 APG X X 21559 APTP X X X 21579 BS.sup.3 X X
22319 BMH X X 21554 BSOCOES X X 21524 DFDNB X X 20047 DIDS X X
21664 DMA X X 20666 DMP X X 20668 DMS X X 22585 DSP X X 21555 DSS X
X 20590 DST X X 20665 DTBP X X 22590 DTBPA X X 21577 DTSSP X X
21550 EADB X X X 21565 EGS X X 23700 FNPA X X X 21560 HSAB X X X
26095 MABI X X X 22310 MBS X X X 27715 NHS-ASA X X X 20669 PNP-DTP
X X X 21552 SADP X X X 21549 SAND X X X 22588 SANPAH X X X 27716
SASD X X X 22325 SIAB X X X X 22320 SMCC X X X 22315 SMPB X X X
21557 SPDP X X X 21556 Sulfo- X X BSOCOES 20591 Sulfo- X X DST
21556 Sulfo- X X EGS 22312 Sulfo- X X X MBS 21553 Sulfo- X X X SADP
22589 Sulfo- X X X SANPAH 22327 Sulfo- X X X SIAB 22322 Sulfo- X X
X SMCC 22317 Sulfo- X X X SMPB 26101 TRAUT'S X X
______________________________________ CROSS-LINKING REAGENTS (part
B) Agent Acronym Chemical Name
______________________________________ ANB-NOS
N-5-azido-2-nitrobenzoyloxysuccinimide APB p-azidophenacyl bromide
APG P-azidophenyl glyoxal APTP n-4-(azidophenylthio)phthalimide
BS.sup.3 bis(sulfosuccinimidyl) suberate BMH bis maleimidohexane
BSOCOES bis[2-(succinimidooxycarbonyloxy)- ethyl]sulfone DFDNB
1,5-difluoro-2,4-dinitrobenzene DIDS
4,4'-diisothiocyano-2,2'-disulfonic acid stilbene DMA dimethyl
adipimidate-2 HCl DMP dimethyl pimelimidate-2 HCl DMS dimethyl
suberimidate-2 HCl DSP dithiobis(succinimidylpropionate) DSS
disuccinimidyl suberate DST disuccinimidyl tartarate DTBP dimethyl
3,3'-dithiobispropionimidate- 2-HCl DTBPA
4,4'-diothiobisphenylazide DTSSP 3,3-dithiobis(sulfosuccinimidyl-
propionate) EADB ethyl-4-azidophenyl 1,4-dithio- butyrimidate EGS
ethylene glycolbis(succinimidyl- succinate FNPA
1-azido-4-fluoro-3-nitobenzene HSAB
N-hydroxysuccinimidyl-4-azidobenzoate MABI
methyl-4-azidobenzoimidate MBS m-maleimidobenzoyl-N-hydroxysulfo-
succinimide ester NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic
acid PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoro- propionate SADP
N-succinimidyl(4-axidophenyl)-1,3'-di- thiopropionate SAND
sulfosuccinimidyl 2-(m-azido-o-nitro-
benzamido)-ethyl-1,3'-dithiopropionate SANPAH
N-succinimidyl-6(4'-azido-2'-nitro- phenyl-amino)hexanoate SASD
sulfosuccinimidyl 2-(p-azidosalicyl-
amido)ethyl-1,3'-dithio-propionate SIAB
N-succinimidyl(4-iodoacetyl)amino- benzoate SMCC succinimidyl
4-(N-maleimidomethyl)- cyclohexane-1-carboxylate SMPB succinimidyl
4-(p-maleimidophenyl)- butyrate SPDP N-succinimidyl
3-(2-pyridyldithio) propionate Sulfo-
bis[2-(sulfosuccinimidooxy-carbonyl- BSOCOES oxy)ethyl]sulfone
Sulfo-DST disulfosuccinimidyl tartarate Sulfo-EGS ethylene
glycolbis(sulfosuccinimidyl- succinate) Sulfo-MBS
m-maleimidobenzoyl-N-hydro-xysulfo- succinimide ester Sulfo-SADP
sulfosuccinimidyl(4-azidophenyldithio)- propionate Sulfo-
sulfosuccinimidyl 6-(4'azido-2'-nithro- SANPAH
phenylamino)hexanoate Sulfo- sulfosuccinimidyl(4-iodoacetyl)amino-
SIAB benzoate Sulfo-SMCC sulfosuccinimidyl 4-(N-maleimido-
methyl)cyclohexane-1-carboxylate Sulfo-SMPB sulfosuccinimidyl
4-(p-maleimido- phenyl)butyrate TRAUT'S 2-iminothiolane-HCl
______________________________________
The cross-linking reagent is applied to the base coat in amounts
such that the desired binding site density is achieved. Binding
site density is that amount of cross-linking reagent, in terms of
moles/g synthetic material, to bind to the base coat while
providing confluent coverage of the surface.
To put the inhibitor in condition for linkage to the base coat, the
cross-linking reagent may be initially coupled separately to both
the base coat and to the inhibitor. The kinetic constants of the
inhibitors are compared before and after coupling to evaluate
effects of the procedure on their kinetic constants. The inhibitor
should remain biologically active after being coupled. Therefore,
standard activity assays specific for the inhibitor to be
immobilized are performed using a standard thrombin solution to
evaluate this capacity.
The protein component of, the base coat may be bound to the
thrombogenesis inhibitor forming a conjugate prior to its adherence
to the synthetic material. The conjugate can then be bound to the
synthetic material as described for base coat binding to achieve
the same effect (TABLE 2). In addition, the thrombogenesis
inhibitor conjugate retains biological activity, and can be used as
an agent to increased half life in the circulation as it is not
easily cleared by the kidney.
SPDP will react with terminal as well as epsilon amino groups,
Since derivatization of a terminal amino group can inactivate a
biologically active protein, T-BLOCK (Pierce Chemical Co.,
Rockford, Ill.) may be used to block that group during
SPDP-derivatization. The T-BLOCK is then removed after
derivatization to restore biological activity.
The invention will be further understood from the following,
non-limiting examples.
EXAMPLE 1: Streptokinase (SK) Immobilization
A. Pretreatment and Activation of Dacron
Dacron polyester 52 (DuPont) is sectioned into 1.0 cm lengths. The
lubricant on and in the woven surface is removed by washing once
for 1 hr with carbon tetrachloride, and twice with 100% CH.sub.3
OH. The methanol is removed by multiple water washes, followed by
one wash in phosphate buffered saline, pH 7.4 (PBS).
The graft material is then subjected to alkaline hydrolysis to
increase available COOH groups. The material is treated with 0.5 M
NaOH at 50.degree. C. for 1 hr. It is then washed with H.sub.2 O
repeatedly, and the following steps initiated immediately.
B. Carbodiimide Derivatization of Activated Dacron
The activated material is placed into 100.0 ml of 10 mM
water-soluble carbodiimide (EDC) in deionized water, pH 4.6-5.0,
for 1 hour at RT with constant stirring. The material is removed
and washed in PBS, to remove excess unbound EDC.
C. Base Coat Formation
The base coat is applied to the lumen of the Dacron graft material.
The derivatized Dacron material is incubated in a 5% HSA solution
in PBS at 1 ml/cm.sup.2 graft material for 24 hr at RT with
constant stirring. The graft is removed and washed in PBS to remove
nonspecifically bound HSA. Approximately 20 mg protein/mg Dacron is
covalently bound.
D. Linkage of SPDP to the Base Coat
The HSA-bound Dacron material is incubated in a 1.0 mM solution of
SPDP in PBS, pH 7.4, to bind SPDP to the HSA (100 mM SPDP/cm.sup.2
base coat). Incubation is terminated after 30-40 min at RT. The
graft is washed with PBS to remove nonspecifically bound SPDP.
E. Activation of SPDP on Base Coat and Measurement of Binding Site
Density
The SPDP-linked material is dried and weighed to obtain its
absolute weight. It is then placed in a 50 mM solution of
dithiotreitol (DTT) for 5 min at RT. This reaction releases
pyridine-2-thione (P-2-T) from the bound SPDP and simultaneously
forms free sulphydryl (SH) groups on the base coat. The released
P-2-T is quantitated by absorption spectrophotometry at 343 nm
using its extinction coefficient (E=8.08.times.10.sup.3), and is
directly proportional to the quantity of bound SPDP or binding
sites. The number of binding sites are calculated and expressed as
moles of sites/g of Dacron.
The material is then washed 5 times in PBS and 4 times in dH.sub.2
O.
F. Linkage of SK to Cross-linker
Albumin-free SK (KabiVitrum, Stockholm, Sweden) is
filter-transfered with use of a PD-10 column (Pharmacia,
Piscataway, N.J.) to remove contaminants and amino acid
preservatives, and to transfer SK into 0.1 M PBS buffer, pH 7.5.
0.5 ml fractions are collected, absorbance values at 280 nm
recorded, and desired fractions pooled. The molar concentration of
pooled SK is determined using its absorptivity coefficient at 280
nm (A.sub.280 1.0%/1.0 cm=7.5). The concentration of pooled SK is
then set to approximately 0.1 mM in PBS buffer. A 20 mM SPDP
solution is prepared in absolute ethanol just prior to use and
mixed with SK in various mole to mole ratios (i.e., 1:10). The
mixture is allowed to incubate for 30 min at 23.degree. C. It is
then filter-transferred into PBS buffer, pH 7.5 using a PD-10
column equilibrated with PBS buffer, pH 7.5. 0.5 ml fractions are
collected, absorbance measurements at 280 nm recorded, and desired
fractions (i.e., A.sub.280 greater than 1.8) pooled. The pool
contains SK linked to 2-pyridine disulphide (SK-2-PD) in PBS
buffer, pH 7.5.
G. Measurement of SPDP Bound to SK
The binding of SPDP to SK can be quantitated by the addition of DTT
which liberates pyridine-2-thione (P-2-T) from SPDP bound to SK,
and which can be measured spectrophotometrically at 343 nm. From
this measurement, the moles of SPDP bound to SK can be calculated.
Each P-2-T released represents one covalent attachment of SPDP to
SK. One mole of SK binds per 1.2 moles SPDP in the present
studies.
H. Linkage of Derivatized SK to Basecoat
The reduced SPDP-linked base coat (having free SH groups) is washed
with PBS to remove the DTT. SPDP-linked SK is then added to the
graft at 50.0 mg/cm.sup.2 Dacron. The solution is incubated
overnight at RT to allow the binding of SPDP-SK to SH groups on the
Dacron graft. The Dacron material with SK covalently immobilized
thereto is then washed and stored in PBS.
I. Immobilized (SK) Activity Analysis
SK, when mixed with human plasminogen forms an active proteolytic
complex that can be quantitated using the chromogenic substrate,
S-2251 (KabiVitrum A.B., Stockholm, Sweden). A standard curve is
constructed using known concentrations of SK (500-10,000 units/ml).
A 60 ml aliquot of each standard is added to 120 ml of 0.2 mM human
plasminogen (American Diagnostica, Greenwich, Conn.), and incubated
at 37.degree. C. for 10 min. SK-plasminogen complex is formed as
well as free plasma which interferes with the analysis. This
interference is eliminated by the addition of 60 ml of 2.0 mg/ml
soybean trypsin inhibitor which inhibits all free plasma. The
quantitative analysis of SK-plasminogen is accomplished by addition
of 420 ml of 0.86 mM S-2251 in 50 mM Tris-HCl, 12 mM NaCl, pH 7.4,
and monitoring the change in absorbance per min at 405 nM at
37.degree. C. The immobilized SK is substituted for the standard
solution in the assay. The measured activity of the material on
S-2251 is then equated to the standard curve.
EXAMPLE 2: Urokinase (UK) Immobilization
A)-H) (Same as described for EXAMPLE 1 except that UK (Abbokinase,
Abbot Chemical Co., Chicago, Ill.) is substituted for SK.)
I. Immobilized UK Activity Analysis
The activity of immobilized UK is evaluated against a standard
curve generated using soluble UK in concentrations of from 10 to
1000 units of UK (refered here as CTA units). The chromogenic
substrate, S-2444 (KabiVitrum AB, Stockholm, Sweden), is used at
0.3 mM to measure activity of UK standards by monitoring change in
absorbance at 405 nm in 50.0 mM Tris, 12.0 mM NaCl, pH 8.8 at
37.degree. C. A standard curve is thus generated. A section of
material with immobilized or bonded UK is placed into the substrate
under the same conditions, and the change in absorbance is
recorded. The activity of the immobilized UK is evaluated by
comparison to the activity of the standard curve.
The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
present embodiments are therefore to be considered in all respects
as illustrative and not restrictive, the scope of the invention
being indicated by the appended claims rather than by the foregoing
description, and all changes which come within the meaning and
range of equivalency of the claims are therefore intended to be
embraced therein.
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